Misfolded protein sensor method in body fluids

ABSTRACT

A catalytic conformational sensor method for detecting abnormal proteins and proteinaceous particles. The method is based on the interaction of a peptide fragment or probe with an abnormal proteinaceous particle. The interaction catalyzes transformation of the probe to a predominately beta sheet conformation and allows the probe to bind to the abnormal proteinaceous particle. This in turn, catalyzes propagation of a signal associated with the test sample-bound probe. As a result signals can be propagated even from samples containing very low concentrations of abnormal proteinaceous particles as is the case in many body-fluid derived samples.

BACKGROUND

1. Field of the Invention

This invention relates generally to a catalytic conformational sensormethod and application of such method for detecting proteins andproteinaceous particles; and more particularly to detecting misfolded ordisease-associated proteins and proteinaceous particles.

2. Related Art

This document claims priority of U.S. provisional patent applications,Ser. No. 60/295,456 filed on May 31, 2001; which is hereby whollyincorporated by reference.

The present invention is not limited to the detection of proteins orpeptides in infectious samples. It also includes detection ofproteinaceous particles such as prions. Prions are small proteinaceousparticles with no nucleic acids, thus are resistant to most nucleic-acidmodifying procedures and proteases. They are infectious particles thatplay key roles in the transmission of several diseases such asCreutzfeldt-Jakob syndrome, transmissible spongiform encephalopathy(TSE), and scrapie a neurological disorder in sheep and goats.¹ Diseasescaused by prions can be hard to diagnose since the disease may be latentwhere the infection is dormant, or may be subclinical where abnormalprion is demonstrable but the disease remains an acute or chronicsymptomless infection. Moreover, normal homologues of a prion-associatedprotein exist in the brains of uninfected organisms, furthercomplicating detection.² Prions associate with a protein referred to asPrP 27-30, a 28 kdalton hydrophobic glycoprotein, that polymerizes(aggregates) into rod-like filaments, plaques of which are found ininfected brains. The normal protein homologue differs from prions inthat it is readily degradable as opposed to prions which are highlyresistant to proteases. Some theorists believe that prions may containextremely small amounts of highly infectious nucleic acid, undetectableby conventional assay methods.³ As a result, many current techniquesused to detect the presence of prion-related infections rely on thegross morphology changes in the brain and immunochemistry techniquesthat are generally applied only after symptoms have already manifestthemselves.¹Clayton Thomas, Tabor's Cyclopedic Medical Dictionary (Phil., F.A.Davis Company, 1989), at 1485.²Ivan Roitt, et al., Immunology (Mosby-Year Book Europe Limited, 1993),at 15.1.³Benjamin Lewin, Genes IV (Oxford Univ. Press, New York, 1990), at 108.

The following is an evaluation of current detection methods.

-   -   Brain Tissue Sampling. Cross-sections of brain can be used to        examine and monitor gross morphology changes indicative of        disease states such as the appearance of spongiform in the        brain, in addition to immunohisto-chemistry techniques such as        antibody-based assays or affinity chromatography which can        detect disease-specific prion deposits. These techniques are        used for a conclusive bovine spongiform encephalopathy (BSE)        diagnosis after slaughter of animals displaying clinical        symptoms. Drawbacks of tissue sampling include belated detection        that is possible only after symptoms appear, necessary slaughter        of affected animals, and results that takes days to weeks to        complete.    -   Prionic-Check also requires liquified-brain tissue for use with        a novel antibody under the Western Blot technique. This test is        as reliable as the immunochemistry technique and is more rapid,        yielding results in six to seven hours, but shares the drawbacks        of the six-month lag time between PrP^(s) accumulation        (responsible for the gross morphology changes) in the brain and        the display of clinical symptoms, along with the need for        slaughter of the animal to obtain a sample.    -   Tonsillar Biopsy Sampling. Though quite accurate, it requires        surgical intervention and the requisite days to weeks to obtain        results.    -   Body Fluids: Blood and Cerebrospinal Sampling. As in the above        detection methods, results are not immediate    -   Electrospray ionization mass spectrometry (ESI-MS), nuclear        magnetic resonance NMR, circular dichroism (CD) and other        non-amplified structural techniques. All of these techniques        require a large amount of infectious sample, and have the        disadvantage of requiring off-site testing or a large financial        investment in equipment.

The difficulty with all of the presently approved tests is that they aretime consuming and are performed POST-MORTEM.

As can now be seen, the related art remains subject to significantproblems, and the efforts outlined above—although praiseworthy—have leftroom for considerable refinement. The present invention introduces suchrefinement.

SUMMARY OF THE DISCLOSURE

The present invention is based on the interaction between lowconcentration levels of abnormal proteinaceous particles and a peptidefragment or probe to induce transformation and propagation of the probebound to the abnormal proteinaceous particles initially present within atest sample. Thus, in a preferred embodiment, infectious levels of atest sample can be propagated even from low concentrations as is thecase in many body-fluid derived samples.

This invention overcomes many of the problems of prior art by usingcatalytic propagation to exploit conformational changes in proteinsassociated with a particular disease process, such as transmissiblespongiform encephalopathy (TSE). Catalytic propagation basicallyamplifies the number of existing protein fragments causing aggregates toform. The aggregates of conformationally changed protein fragments arethen easily detected using common analytical techniques. As a result,the present invention allows testing to be done using rapid andcost-effective analytical techniques, even on, heretofore difficult todetect, small sample sizes and is widely applicable to tissues and bodyfluids other than those found in brain. The invention is also relativelynoninvasive in that it does not need to be performed post-mortem.

Moreover, results can easily and immediately interpreted using familiaranalytical instrumentation. Additionally, the present invention canamplify a weak signal, thus can be successfully applied to small or weaksamples such as those associated with body fluids; thereby opening thedoor to analysis of tissues and fluids for the elusive diseasesdiscussed above.

All of the foregoing operational principles and advantages of thepresent invention will be more fully appreciated upon consideration ofthe following detailed description, with reference to the appendeddrawings, of which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pictoral representation of conformers of transmissiblespongiform encephalopathies (TSE);

FIG. 2 is a pictoral representation of TSE protein detection schema;

FIG. 3 is a graph showing the conformational changes associated with apoly-L-lysine test peptide using circular dichroism;

FIG. 4 is a graph comparing the circular dichroism results of thepoly-L-lysine test peptide at different temperatures and pH;

FIG. 5 is a table comparing the circular dichroism results of thepoly-L-lysine test peptide at different temperatures and pH;

FIG. 6 is a graph of data for fluorescence resonance energy transfer(FRET) experiments for proximal and distal locations in an α-helicalbundle structure undergoing conformational change; and

FIG. 7 is a graph of the driving force necessary to overcome the energydifference between two different conformational states.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention detects the presence of abnormal proteins andproteinaceous particles based on a method that utilizes catalyticpropagation. Upon interaction of a sample, containing abnormal proteinsor proteinaceous particles, with a peptide probe of the invention, thepeptide probe undergoes conformational changes resulting in theformation of aggregates. The addition of the abnormal proteins andproteinaceous particles catalyzes the formation of the aggregates andcauses further propagation of this conformational transition. Theresulting aggregates are then easily detected using common analyticalinstrumentation and techniques.

The abnormal proteins and proteinaceous particles on which the inventionfocuses are proteins, protein based chemical structures such as prionsand protein subunits such as peptides that are capable of conformationalchanges that lead to the formation of aggregates and ultimately todisease states.

These proteins and proteinaceous particles form aggregates by shiftingfrom a monomeric to a multimeric state. The shift from one distinctstate to the other requires a driving force that is commensurate withthe energetic difference between the two conformational states as shownin FIG. 7.

A preferred example of such proteinaceous particles is that of a prionprotein. Prions can exist in one of two distinct conformationscharacterized by having a secondary protein structure that is eitherpredominately alpha-helical or predominately beta-sheet; where thepredominately beta-sheet conformation has a much higher preference toexist in a multimeric state. As a result, predominately beta-sheet (orbeta rich) secondary structure is more typical of abnormally folded ordisease-causing proteinaceous particles since their preference toaggregate is likely to be disruptive in an in vivo environment.

FIG. 1 shows illustrations of both the alpha-helical monomer 10 and thebeta-sheet dimer 12 forms of a TSE conformer. The normal wild-type (wt)form of prion protein (PrP^(c)) prefers a monomeric state, while theabnormal disease-causing form (PrP^(Sc)) more readily takes on amultimeric state.

This distinction between the secondary structure of the normal form ofprion protein and the abnormal form as well as its propensity to causeaggregation is exploited in the present invention to allow detection ofthe abnormal form even in samples with very low levels of infectiousabnormal protein.

The mechanism of the invention is shown in a schematic in FIG. 2. Thetop row of the schematic shows an example of an unknown sample of TSEprotein represented as containing beta-sheets 12. The beta-sheets arethen disaggregated by subjecting the sample to commonly knowndisaggregation methods such as sonication. This is followed by theaddition of labeled peptide probes 14 which are allowed to bind to thesample 12. Presence of the beta-sheet conformation in the sample 12induces the peptide probes to also shift to beta-sheet formation 16. Inthis manner the transition to beta-sheet is propagated among the peptideprobes 14 thereby causing new aggregates 18 to form. The resultingtransition to a predominately beta-sheet form and amplified aggregateformation can then easily be detected using common analytical techniquessuch as light scattering and circular dichroism (CD); and in aparticularly preferred embodiment where the peptide probe is fluorescentlabeled, fluorescence detection instrumentation can also be used.

The bottom row of FIG. 2 shows an alternative example in which theunknown sample of TSE protein is represented in its normal alpha-helicalform 10. For consistency, the sample is subjected to the samedisaggregation process described above. Upon addition of the labeledpeptide probes 14, neither a transition to beta-sheet form nor bindingto the unknown samples occurs. As a result, there is no aggregatefluorescence signal in the case of a labeled peptide probe as well as nodetection of aggregate formation by other analytical tools. Based onthis schematic, unknown samples can be tested for the presence orabsence of such abnormal protein conformations or sequences.

A preferred embodiment of the invention involves the following basicprocedures. Peptide probes 14 are selected in order to be added to anunknown or test sample 20 at a later stage in the process. The peptideprobes 14 are preferably proteins or peptide sequences that havesecondary structures of predominately alpha-helix or random coil. In aparticularly preferred embodiment, the peptide probes 14 are peptidefragments consisting of a helix-loop-helix structure as found in lysine.In another particularly preferred embodiment, the peptide probes can bemade of a peptide sequence chosen from wild-type (wt) TSE, from adesired species-specific TSE peptide sequence, or even from aselectively mutated TSE sequence that has been mutated in such a manneras to render it destabilized and noninfectious. Additionally, extrinsicfluors such as pyrene can be added or designed into the peptide probe toallow detection of anticipated conformational changes using commonfluorescence detection techniques.

Once a peptide probe 14 is selected, it is added to a test sample 20.Prior to the addition of the peptide probe 14, however, it is preferredto have the sample 20 subjected to disaggregation techniques commonlyknown in the art, such as sonication. The disaggregation step allows anypotentially aggregated sample material 20 to break apart so that thesedisaggregated sample materials 22 are more free to recombine with thenewly introduced peptide probes 14; thereby facilitating the anticipatedcatalytic propagation.

After the test sample 20 or disaggregated test sample 22 is allowed tointeract with the peptide probes 14. The resulting mixture is thensubjected to analytical methods commonly known in the art for thedetection of aggregates and to fluorescence measurements in cases wherefluorescent peptide probes 14 are used.

Unknown or test samples 20 containing any dominant beta-sheet formationcharacteristic of abnormally folded or disease-causing proteins resultsin an increase in beta-sheet formation and consequently aggregateformation in the final mixture containing both the test sample 20 andthe peptide probes 14. Conversely, unknown or test samples 20 which lacka predominantly beta-sheet secondary structure will neither catalyze atransition to beta-sheet structure 16 nor will propagate the formationof aggregates 18.

One of ordinary skill in the art can appreciate that the means by whichthe initial conformational change can be triggered in the test samples20 can be varied as described in the following examples. The binding ofa metal ligand could direct a change in the protein scaffolding andfavor aggregation. The expression or cleavage of different peptidesequences can promote advanced aggregation leading to fibril and plaqueformation. Genetic point mutations can also alter the relative energylevels required of the two distinct conformations, resulting in midpointshifts in structural transitions. Furthermore, an increase inconcentration levels could be sufficient to favor the conformationaltransition. Regardless of the initial trigger mechanism, however, thedisease process in many of the abnormal protein conformations such as inprion-related diseases always involves the catalytic propagation of theabnormal conformation, resulting in transformation of the previouslynormal protein.

One of ordinary skill in the art can also appreciate that there are manycommon protein aggregate detection techniques many of which are based onoptical measurements. These optical detection techniques include, butare not limited to, light scattering, or hydrophobicity detection usingextrinsic fluors such as 1-anilino-8-napthalene sulfonate (ANS) or CongoRed stain, fluorescence proximity probes on the peptide fragments,including fluorescence resonance energy transfer (FRET) & quenching ofintrinsic tryptophan fluorescence through either conformational changeof monomer or binding at interface in alpha-beta heterodimer; theN-terminal loop region is particlularly interesting in this regardselective binding to target protein, circular dichroism (CD) monitoringof actual conformation, nuclear magnetic resonance (NMR). Otherdetection techniques include equilibrium ultracentrifugation orsize-exclusion chromatography at the aggregation stage as well as otherstructural techniques. Many of these enumerated optical and structuralmethods are rapid, cost-effective and accurate.

Experiments were performed using model systems to show theconformational changes involved in the transition from a predominatelyalpha-helix to a beta-rich form. The model systems chosen used readilyavailable, nonneurotoxic polyamino acids such as polylysine andpolyglutamine. The polyamino acids were chosen because of theiravailability and more importantly because they are safe to handle thuseliminating the need for special handling or donning cumbersome extraprotective gear.

FIG. 3 shows a circular dichroism graph of experimentation withpoly-L-lysine 20 micro Molar (μM) 52,000 molecular weight (MW) as apeptide probe. The resulting graphs show:

-   -   Sample 24 which was maintained at pH7, 25° C. resulting in a        minimum at approximately 205 namometers (nm) indicating random        coil structure.    -   Sample 26 which was maintained at pH11, 50° C. resulting in a        minimum at approximately 216 namometers (nm) indicating        beta-sheet structure.    -   Sample 28 which was a 1:1 combination of samples maintained at        pH7, 25° C. and at pH11, 50° C. resulting in a minimum at        approximately 216 namometers (nm) indicating beta-sheet        structure.    -   Sample 30 which was a 1:1 combination of samples maintained at        pH7, 50° C. and at pH11, 50° C. resulting in a minimum at        approximately 216 namometers (nm) indicating beta-sheet        structure.

FIG. 4 shows an absorbance graph of experimentation with poly-L-lysine70 mircomolar (μM) 52,000 molecular weight (MW) as a peptide probe. Theresulting graphs show:

-   -   Sample 32 which was maintained at pH11, 25° C. resulting in a        plateau at approximately 0.12 indicating predominately        alpha-helical structure.    -   Sample 34 which was maintained at pH7, 50° C. resulting in a a        plateau at approximately 0.22 indicating random coil structure.    -   Sample 36 which was a 10:1 combination of samples maintained at        pH7, 50° C. and at pH11, 50° C. resulting in a steeper incline        from approximately 0.22 to 0.33 indicating an accelerated        transition from random coil to beta-sheet structure.    -   Sample 38 which was a 10:1 combination of samples maintained at        pH7, 25° C. and at pH11, 50° C. resulting in a gradual incline        from approximately 0.22 to 0.26 indicating a transition from        random coil to beta-sheet structure.

FIG. 4 shows general circular dichroism results of experimentation withpoly-L-lysine at varying temperatures and pH indicating its potentialfor transitioning from random coil to beta-sheet under the varyingenvironmental conditions. The results indicate that both temperature andpH play an important role in the transition.

The observations based on all of the modeling experimentation describedabove show that the addition of a relatively small amount of beta-sheetpeptide to random coil sample can result in a shift towards a beta-richconformation and such changes can be accelerated depending on thetemperature and pH environment of the samples.

FIG. 6 shows experimentation results using pyrene as a fluorescent probein proximal and distal locations in an alpha-helical bundle structureundergoing conformational change. The pyrene excimer formation 42 isshown at 480 nm and the spectra for a predominately alpha-helicalstructure 40 is contrasted as well. Those skilled in the art wouldappreciate that other fluorescent probes such as Fourier TransformInfrared Spectroscopy (FITC) can also be used.

A primary objective of this invention also encompasses use of thecatalytic propagation of conformational change to directly correlate themeasures of abnormal prion presence with levels of infectivity. For thisreason we favor implementation of the invention in a manner where thereis no increase in resulting infectious products as a result of thepropagation. This can be achieved by placing a “break” in the linksbetween the chain of infection, transmission and propagation of theabnormal form. Such a “break” must occur at the transitional stagebetween the dimer and multimer forms of the aggregate. The physicalformation of the multimer form can be blocked by simply impeding thestep which leads to its formation. This may be done, preferably by usinga large pendant probe or by a neutral “blocker” segment, bearing in mindthat probes on linkers or “tethers” are more likely to encounter eachother and thus result in amplifying the signal.

Furthermore, it follows inherently in everything that is prescribed inthe teachings of the provisional that in the practice of this invention,neither the peptide probe nor the final mixture is infectious—unlike allother prior art in the field of prion assay.

All of the foregoing information is found within the aforementionedprovisional patent application Ser. No. 60/295,456 filed May 31, 2000from which priority is claimed. Although not included in the provisionalapplication, analytical methods for appraising aggregation of proteinsare included in the following publications which are prior art.Freifelder, David. Physical Biochemistry: Applications to Biochemistryand Molecular Biology, (W. H. Freeman Press, New York, 2nd ed. 1982).Copeland, Robert. Analytical Methods for Proteins, (American ChemicalSociety Short Courses 1994). both of which are wholly incorporatedherein as prior art.

Accordingly, the present invention is not limited to the specificembodiments illustrated herein. Those skilled in the art will recognize,or be able to ascertain that the embodiments identified herein andequivalents thereof require no more than routine experimentation, all ofwhich are intended to be encompassed by claims.

Furthermore, it will be understood that the foregoing disclosure isintended to be merely exemplary, and not to limit the scope of theinvention—which is to be determined by reference to the appended claims

1.-20. (canceled)
 21. An isolated peptide reagent that interactspreferentially with pathogenic forms of a conformational disease proteinas compared to nonpathogenic forms of the conformational diseaseprotein.
 22. The peptide reagent of claim 21, wherein the peptidereagent includes the amino acid sequence (G)n, where n=1, 2, 3 or 4, atthe N-terminal end, at the C-terminal end, or at both the N-terminal andC-terminal end.
 23. The peptide reagent of claim 21, wherein the peptidereagent is genetically encoded.
 24. A polynucleotide encoding a peptidereagent according to claim
 23. 25. A composition comprising thepolynucleotide of claim
 24. 26. The peptide reagent of claim 21, whereinthe conformational disease is a prion-related disease, the pathogenicprotein is PrP^(Sc), and the nonpathogenic form is PrP^(C).
 27. Thepeptide reagent of claim 26, wherein the peptide reagent is derived froma fragment of a prion protein.
 28. A composition comprising a peptidereagent according to claim
 26. 29. A complex comprising the peptidereagent of claim 26 and a pathogenic prion protein.
 30. A peptide havinga predominantly alpha-helix secondary structure, random coil secondarystructure, or a combination thereof, that interacts with misfoldedproteinaceous particles.
 31. The peptide of claim 30, wherein themisfolded proteinaceous particles are PrP^(SC) particles.
 32. Thepeptide of claim 30, wherein the peptide undergoes a conformationalshift that results in a decrease in alpha-helix and/or random coilsecondary structure and an increase in beta-sheet secondary structureupon contact with misfolded proteinaceous particles or upon contact withanother such peptide that has undergone such a conformational shift. 33.The peptide of claim 30, wherein the peptide has a helix-loop-helixstructure.
 34. The peptide of claim 30, wherein the peptide comprises asequence found in a wild-type transmissibile spongiform encephalopathy(TSE) peptide sequence, a species-specific TSE peptide sequence, or amutated TSE sequence mutatet to be destabilized and/or noninfectious.35. The peptide of claim 30, wherein the peptide is labeled with adetectable label.
 36. The peptide of claim 30, wherein the peptide isnon-infectious.
 37. A composition comprising a peptide of claim 30 boundto a misfolded proteinaceous particle.
 38. A composition comprising apeptide of claim
 30. 39. The composition of claim 38, wherein themisfolded proteinaceous particle is a PrP^(SC) particle.
 40. A methodfor detecting the presence of a pathogenic prion in a sample comprising:(a) contacting a sample suspected of comprising a pathogenic prion witha first peptide reagent according to claim 26 under conditions thatallow binding of the first peptide reagent to the pathogenic prionprotein, if present; and (b) detecting the presence the pathogenicprion, if any, in the sample by its binding to the first peptidereagent.
 41. The method of claim 40 wherein the first peptide reagent isdetectably labeled.
 42. A method for detecting the presence of apathogenic prion in a sample comprising: (a) contacting a samplesuspected of comprising a pathogenic prion with a first peptide reagentaccording to claim 26 under conditions that allow interaction of thefirst peptide reagent with the pathogenic prion protein, if present; and(b) detecting the presence the pathogenic prion, if any, in the sampleby its interaction with the first peptide reagent.
 43. A method fordetecting the presence of a pathogenic prion in a sample comprising: (a)contacting a sample suspected of comprising a pathogenic prion with afirst peptide reagent according to claim 30 under conditions that allowinteraction of the first peptide reagent to the pathogenic prionprotein, if present; and (b) detecting the presence the pathogenicprion, if any, in the sample by its interaction with the first peptidereagent.